Due to rising rates of resistance, policy and opinion leaders have called for the development of entirely new paradigms of antimicrobial therapy to complement traditional antibiotics. Here we propose an innovative adjunctive antimicrobial approach to both prevent emergence of resistance in lethal pathogens and potentially treat XDR/PDR pathogens. We have found that recombinant human transferrin (rhTransferrin) effectively sequestered iron from Acinetobacter baumannii, methicillin resistant Staphylococcus aureus (MRSA), and Candida albicans, inhibiting their growth. Treatment with rhTransferrin improved survival of mice infected with lethal inocula of all three pathogens. Furthermore, adjunctive rhTransferrin therapy markedly reduced emergence of rifampin-resistance during treatment of mice with MRSA bacteremia. Finally, most recently, we found in vitro synergy between rhTransferrin and ciprofloxacin (cipro) against cipro-resistant K. pneumoniae. We will test the generalizability of these findings against Klebsiella pneumoniae and A. baumannii. Our novel strategy is to treat infection and inhibit emergence of resistance by rendering the host environment inhospitable to microbial growth. We will optimize this strategy as adjunctive therapy for infections in 3 AIMS: AIM 1: Determine the in vitro effects of adjunctive rhTransferrin on susceptibility and emergence of resistance to key antibacterial agents used to treat highly resistant Gram negative pathogens. The impact of rhTransferrin on in vitro susceptibility and resistance emergence to ciprofloxacin or meropenem will be defined for a panel of genetically well-characterized K. pneumoniae and A. baumannii strains. Trace metals and heme will be added back to determine if they reverse transferrin-mediated effects. In vitro time kill assays wil be used to determine how rhTransferrin alters the kinetics of antibacterial killing of the microbes AIM 2: Define optimal pharmacokinetics and pharmacodynamics of antibacterial agents with versus without adjunctive rhTransferrin. A sophisticated chemostat system will define dosing to optimize pharmacokinetic-pharmacodynamic (PK-PD) impact of rhTransferrin and antibiotic-transferrin combination therapy on cidality and emergence of antibiotic resistance of K. pneumoniae and A. baumannii. PD will be evaluated by measuring rhTransferrin-mediated metal sequestration and its impact on microbes. AIM 3: Define the in vivo efficacy and prevention of resistance of adjunctive rhTransferrin therapy. Aims 1 and 2 will inform dosing strategies to define efficacy of adjunctive rhTransferrin plus antibiotics vs. antibiotic alone in mice infected iv or via the lung with K. pneumoniae or A. baumannii. Some groups will be treated with trace metals or heme to determine if efficacy is reversed. IMPACT: rhTransferrin already has been studied in clinical trials of patients with iron overload. Completion of the proposed studies will enable rapid clinical translation of rhTransferrin as adjunctive therapy to prevent the emergence of antibacterial resistance among pathogens with a high propensity to develop resistance.